The use of 32P-postlabelling in studies of the nature and origin of DNA adducts formed by bile from patients with familial adenomatous polyposis and from normal patients.

32P-postlabelling is a highly sensitive technique for the detection of DNA adducts. It is unique in that it requires no prior knowledge of the nature of adducts or adduct-forming species under investigation. In the past, we have used this technique to investigate the role of bile in the production of foregut adenomas in patients with familial adenomatous polyposis (FAP). We have found that bile contains constituents that form DNA adducts directly, and after metabolic activation, and that the bile of FAP patients has an increased capacity for adduct formation with DNA in vitro, in human cell lines in culture, and in the gastrointestinal tract of rats given bile by gavage. The sensitivity of 32P-postlabelling is such that it is difficult to obtain sufficient quantities of DNA adducts for chemical analysis. The nature of the adducts produced by bile, or of the bile constituents that produce them is as yet undetermined. In the present studies, we have combined 32P-postlabelling with indirect methods to gain some insight into the nature of DNA adducts produced by bile and the properties of the reactive species that form them. Firstly, bile was incubated with synthetic monodeoxynucleotides or polydeoxynucleotides. Bile did not produce adducts when incubated with monodeoxynucleotides or single-stranded polydeoxynucleotides. However, it did produce adducts when incubated with double-stranded polydeoxynucleotides. The pattern of adduct formation suggested that human bile forms a mixture of adenine and guanine adducts. Secondly, bile was fractionated by extraction with blue cotton or with neutral, acid or alkaline organic solvent. Blue cotton, which efficiently and selectively absorbs mutagens having 3 or more fused aromatic rings, did not absorb biliary constituents that could form adducts with DNA in vitro or with DNA of MCL-5 cells, a metabolically competent human cell line. This suggests that biliary DNA adduct precursors are polar compounds that contain fewer than 3 aromatic rings or are non-aromatic. Acidic organic extracts of human bile produced much higher levels of DNA adducts in vitro or with DNA of MCL-5 cells than did neutral or alkaline organic extracts, suggesting that constituents of bile that form DNA adducts are acidic in nature.

Bile acids do not form adducts when incubated with DNA in vitro.

Bile acids have been implicated in the aetiology of colon cancer. We have previously found, using 32P-postlabelling, that bile from control patients and from patients with familial adenomatous polyposis (FAP) produces DNA adducts when incubated with salmon sperm DNA in vitro. In the present study we have incubated the common primary and secondary, conjugated and unconjugated bile acids with salmon sperm DNA in vitro, in both the presence and absence of metabolic activation (Aroclor-induced rat liver S9). The DNA was then purified and assayed for the formation of DNA adducts using the nuclease P1 method of 32P-postlabelling. Under the conditions of the assay none of the bile acids tested with or without metabolic activation produced any evidence of DNA adduct formation. It is therefore unlikely that the adduct-forming ability of human bile can be attributed to bile acids or their metabolites.

Appearance of artefacts when using 32P-postlabelling to investigate DNA adduct formation by bile acids in vitro: lack of evidence for covalent binding.

We reported (Scates et al. Carcinogenesis 1994, 15, 2945-2948) that incubating a range of bile acids with DNA in vitro, with or without exogenous metabolic activation, gave no evidence of DNA adduct formation as judged by the nuclease P1 method of 32P-postlabelling. In contrast Hamada et al. (Carcinogenesis 1994, 15, 1911-1915), also using postlabelling, claimed that chenodeoxycholic acid, lithocholic acid, glycolithocholic acid and taurolithocholic acid bound covalently to DNA in vitro. To investigate this discordance we incubated solutions of salmon sperm DNA for 1 h at 37 degrees C with 1 mg/ml of cholic acid, chenodeoxycholic acid, lithocholic acid, glycolithocholic acid or taurolithocholic acid. Each incubate was extracted extensively with diethyl ether after which a sample of DNA was taken and 32P-postlabelled using the nuclease P1 method. The DNA in the remaining incubate was precipitated from high salt solution with ethanol. Aliquots of this DNA were postlabelled. The remainder of the DNA was purified with proteinase-K, ribonuclease, phenol-chloroform, precipitated and postlabelled. Parallel incubates were made with the same bile acids, under the same conditions but in the absence of DNA and were then extracted, precipitated and postlabelled as described above. When DNA was present in the incubate but was not precipitated, chenodeoxycholic acid, lithocholic acid, glycolithocholic acid and taurolithocholic acid, but not cholic acid, produced spots similar to those reported by Hamada et al. No such spots were seen when DNA was postlabelled after precipitation, or after precipitation and purification. These same bile acids produced spots when postlabelled in the absence of DNA, but spots were absent when these incubates were precipitated and purified before postlabelling. We conclude that the spots obtained when bile acids are incubated with DNA which is not precipitated from high salt before it is postlabelled are technical artefacts, and cannot be regarded as evidence that bile acids bind covalently to DNA to form adducts. We also confirm reports (Vulimiri et al. Carcinogenesis 1994, 15, 2061-2064) that bile acids alone can produce spots when incubated with T4 polynucleotide kinase and [gamma-32P]ATP.

DNA adducts, detected by 32P-postlabelling, in the foregut of patients with familial adenomatous polyposis and in unaffected controls.

Duodenal neoplasms in patients with familial adenomatous polyposis (FAP) cluster around the ampulla; duodenal neoplasia is common but gastric neoplasia is rare. These observations suggest that bile enhances neoplasia by carrying carcinogens to target cells. A critical step in carcinogenesis is the reaction of carcinogens with DNA to form chemical adducts. Using 32P-postlabelling to measure DNA adducts we asked: (i) are there more adducts in the duodenum of patients with FAP than in the duodenum of normal patients? (ii) Are there more adducts in the duodenum than in the stomach? We measured adducts in duodenal biopsies from 51 patients with FAP and 30 age-matched controls; and paired gastric and duodenal biopsies from 31 FAP patients and six controls. The foregut of 90% of all patients studied contained DNA modifications with characteristics of aromatic non-polar DNA adducts. In duodenal biopsies, adduct labelling per 10(9) DNA nucleotides was significantly higher in FAP patients (median 15.0, range 0-162) than in normal patients (median, 7.5, range 0-40; P = 0.0002). In paired duodenal and gastric biopsies from 31 FAP patients, adduct labelling was significantly higher in duodenal DNA (median 15, range 0-109) than in stomach DNA (median 8.0, range 0-40; P = 0.0004). Age, gender or smoking did not account for these differences. Gastric adducts (median 3.5, range 0-10) were lower than duodenal adducts (median 8.5, range 0-29) in paired biopsies from six control patients, P = 0.031). These results support the hypothesis that pancreaticobiliary secretions may be involved in the pathogenesis of foregut neoplasia in FAP.

DNA adducts, detected by 32P-postlabelling, in DNA treated in vitro with bile from patients with familial adenomatous polyposis and from unaffected controls.

Patients with familial adenomatous polyposis (FAP) have a high risk of developing duodenal adenomas and carcinomas. The distribution of these neoplasms resembles mucosal exposure to bile, suggesting that bile may play a role in adenoma development. Our previous results, using DNA adducts detected by 32P-postlabelling as an index of genotoxicity, have supported this hypothesis. We found significantly higher adduct labelling in the duodenum of FAP patients than in the duodenum of control patients and significantly higher labelling in the small bowel of rats gavaged with FAP bile than in rats given control bile. We have now investigated the ability of human bile to form adducts with DNA in vitro. Bile obtained from the gallbladder of 18 FAP patients immediately before colectomy, and from 18 control patients, was incubated with salmon sperm DNA in solution at 37 degrees C for 1 h, after which the purified DNA was analysed for DNA adducts, using the nuclease P1 method of 32P-postlabelling. Relative adduct labelling values (RAL, adducts per 10(9) nucleotides) produced by FAP bile samples were significantly higher than RAL values produced by control bile samples (medians 197 versus 86, P = 0.0016, Mann-Whitney test). We found a consistent pattern of adduct labelling, varying in intensity between samples. Adduct spots were eluted from TLC plates and analyzed by reverse-phase HPLC. Each major spot gave several peaks that were consistent between bile samples from different patients and were similar in FAP and control bile. These results indicate that bile from FAP and control patients contains similar, directly acting genotoxic compounds but that levels are higher in FAP than in control patients. This suggests that bile from FAP patients is more genotoxic than bile from control patients. Incubation of bile with free-radical scavengers and deconjugating enzymes failed to influence adduct labelling in this system.

32P-postlabelling studies of target tissues and bile from patients with familial adenomatous polyposis and from unaffected controls.

Patients with the inherited form of colon cancer, familial adenomatous polyposis (FAP), are at high risk of developing duodenal adenomas and carcinomas. The periampullary clustering of these neoplasms suggests that bile plays a role in their development. We investigated this theory using 32P-postlabelling to detect DNA adducts. We found significantly higher adduct levels in duodenum of FAP patients than in unaffected controls, and higher levels in duodenum than stomach. Levels of adducts were significantly higher in the small bowel of rats gavaged with FAP gallbladder bile than in the small bowel of those that received control gallbladder bile. We found that bile from FAP gallbladder produced significantly more DNA adducts than control gallbladder bile when incubated with salmon sperm DNA in vitro. These results support the hypothesis that the bile of FAP patients may be involved in the development of duodenal adenomas in these patients.

DNA adducts detected by 32P-postlabelling, in the intestine of rats given bile from patients with familial adenomatous polyposis and from unaffected controls.

Duodenal adenomas are common in patients with familial adenomatous polyposis (FAP) and cluster around the papilla, suggesting the involvement of bile in their development. Using 32P-postlabelling we determined levels of DNA adducts in the small bowel and colon of rats treated with bile from FAP and control patients. We found a significantly higher level of adducts in the small bowel of rats treated with FAP gallbladder bile compared with control gallbladder bile (P = 0.0034). This result supports the hypothesis that bile plays a role in the development of neoplasia in the foregut of FAP patients.

Detection of telomerase activity in human prostate: a diagnostic marker for prostatic cancer?

OBJECTIVE: To assess the role of telomerase activity as a marker for the development of prostate cancer in men with existing benign prostatic hyperplasia (BPH), a known risk factor for prostatic carcinoma. MATERIALS AND METHODS: Telomerase activity was assayed, using a highly sensitive polymerase-chain reaction-based assay, in nine biopsies from patients with prostatic cancer, 16 from patients clinically diagnosed with BPH and 11 from patients with no evidence of prostatic disease. RESULTS: Telomerase activity was detectable in eight of the nine prostate cancer biopsies, in none of the normal prostates and in six of the 16 BPH biopsies. CONCLUSION: The finding of telomerase activity in six of 16 biopsies from patients with BPH could indicate early prostate cancer and suggests that telomerase activity may be of use as a biomarker in patients diagnosed with BPH and who may subsequently develop prostate cancer.

High pH reduces DNA damage caused by bile from patients with familial adenomatous polyposis: antacids may attenuate duodenal polyposis.

Patients with familial adenomatous polyposis (FAP) develop periampullary duodenal tumours, suggesting that bile contributes to their formation. The hypothesis that bile contains carcinogens has been tested by looking for DNA adducts (markers of carcinogen exposure) in the duodenum of patients with or without FAP and by determining whether bile can produce DNA adducts in vitro. Using 32P-postlabelling to detect adducts, there was an excess (compared with unaffected patients) of DNA adducts in the duodenum of FAP patients and an excess of DNA adducts in the small bowel of rats treated with FAP bile, while bile from FAP patients formed significantly more DNA adducts in vitro than did bile from controls. In this study it is shown that the excess of adduct labelling produced by FAP bile in vitro depends on the pH of the incubate. While adduct labelling at pH 6-8 did not differ significantly between bile from six FAP patients and six controls, at pH 4-5 FAP bile, but not control bile, produced a near threefold excess of adduct labelling over that at pH 6-8. Therapy that increases duodenal pH may therefore alleviate duodenal polyposis.

Differences in the levels and pattern of DNA-adduct labelling in human cell lines MCL-5 and CCRF, proficient or deficient in carcinogen-metabolism, treated in vitro with bile from familial adenomatous polyposis patients and from unaffected controls.

In patients with familial adenomatous polyposis (FAP), duodenal adenomas cluster around the ampulla and their distribution closely resembles mucosal exposure to bile, suggesting a role for bile in their development. Previous studies using 32P-postlabeling to detect DNA adducts, have provided evidence to support this hypothesis. We have now investigated the role of metabolic activation in influencing the levels and patterns of adduct formation by incubating precolectomy gallbladder bile from FAP patients and bile from unaffected controls with human lymphoblastoid cell lines that are metabolically proficient (MCL-5), or deficient (CCRF). 32P-Postlabeling assays showed that MCL-5 cells (genetically engineered to express five human cytochromes P450 and microsomal epoxide hydrolase) formed characteristic adduct spots with benz[a]pyrene, benzo[g]chrysene, 7,12-dimethylbenz[a]anthracene, benzidine, sterigmatocystin and 3-methylcholanthrene, whereas CCRF cells did not. Accordingly, we assayed the ability of bile from FAP patients and controls to form DNA adducts in MCl-5 and in CCRF cells. Relative adduct labelling (RAL) in MCL-5 cells treated with FAP bile (12 patients, median 10, range 1-74) was significantly higher than in cells treated with control bile (12 patients, median 4, range 0-9; P = 0.0007) as was RAL for the two major adduct spots. These two major adduct spots were not observed when bile was incubated with CCRF cells. The adduct spots in CCRF DNA appeared in positions similar to some of the minor adduct spots produced by bile in MCL-5 DNA and to some of the adduct spots seen previously when bile was incubated with salmon sperm DNA in vitro. RAL for CCRF cells incubated with FAP bile (seven patients, median 23.0, range 0-49) was significantly higher than in cells treated with control bile (seven patients, median 2.0, range 0-26; P = 0.0034). These results indicate that the bile obtained from FAP and control patients contains adduct-forming substances, some of which are direct acting and some of which require metabolic activation. In both cell lines, FAP bile produced significantly higher adduct labelling than control bile, adding to the evidence that bile can induce DNA damage in vitro and plays a role in neoplastic development in the FAP foregut.

Lack of telomerase in desmoids occurring sporadically and in association with familial adenomatous polyposis.

BACKGROUND: Telomerase activity may be required for unlimited growth of cells and is repressed in most somatic tissues, but is detectable in immortal cell lines, germ cells, many malignancies and some benign lesions. Desmoids are proliferative, locally invasive, non-metastasizing fibromatous tumours which rarely regress. They occur frequently in familial adenomatous polyposis (FAP), causing significant morbidity and death. Telomerase activity was assayed in desmoids from patients with and without FAP to assess the role of telomerase in the development of these lesions, and its potential as a prognostic marker and possible target for treatment. METHODS: Protein extracts from 11 desmoids from nine patients with FAP, and ten desmoids from ten patients without FAP, were analysed for telomerase activity by the telomeric repeat amplification protocol, a sensitive polymerase chain reaction-based assay. Six fibrosarcomas and a fibrosarcoma cell line were used as positive controls; all displayed telomerase activity. RESULTS: No telomerase activity was detected in any of the 21 desmoids studied. CONCLUSION: These results indicate that desmoid tumours are one of the intriguing exceptions to the emerging view that re-expression of telomerase activity accompanies the development of preneoplastic and neoplastic tissues, and suggest that alternative mechanisms may operate in these proliferative neoplasms.